Although the prototypes of breast cancer suppressor genes, BRCA1 and BRCA2, have been identified, the genetic defects responsible for the majority of breast cancer remain elusive. In addition, essentially all cancer suppressor genes identified are autosomal. X-linked tumor suppressors are of great interest as one allele of these genes can be silenced by X-chromosome inactivation in women. The X-linked FoxP3 is a member of the forkhead/winged helix transcription factor family. Mutations of FoxP3 in both mice (FoxP3sf) and man result in severe autoimmune disease with early lethality. We have recently made a surprising observation that FoxP3sf/+ mice develop cancer at a high rate. The majority of the cancers are mammary carcinomas that have silenced FoxP3 but have over-expressed ErbB2. The overall goal of the project is to build on this exciting preliminary study, to reveal the significance of FOXP3 as a cancer suppressor and to understand the mechanisms by which FoxP3 control breast cancer development in the mouse and in humans. We will continue our effort to study the significance of FoxP3-mediated repression of HER-2/ErbB2 suppression in the mouse model of breast cancer. In addition to HER-2/Neu, our preliminary data also suggest Skp2 and CD24 as novel FoxP3 targets. Their relevance in mammary cancer formation will be tested in vitro 2-D and 2-D culture as well as in vivo in transgenic and knock out models. In addition, we will take a global approach to identify target genes regulated by the FOXP3 protein. Furthermore, we have found widespread down- regulation of the FOXP3 gene among human breast cancer samples. We will test the hypothesis that the two alleles of the FOXP3 locus are silenced by loss of heterozygocity (LOH) in conjunction with X-chromosomal inactivation. In those samples that we have failed to detect mutation or LOH, we will test the possibility that bi-allelic epigenetic mechanisms may be involved in silencing the FOXP3 locus. Finally, we will determine expression of HER-2, SKP2, CD24 and other potential FOXP3 targets among the human breast cancer samples in order to establish a spectrum of mechanisms by which FOXP3 controls breast cancer development. Our proposed studies will not only have the potential for establishing FOXP3 as an important breast cancer suppressor gene but also provide a genetic mechanism for the abnormal expression of the major breast cancer oncogene, HER-2, in humans as well as a molecular mechanism for a major therapeutic modality.